Abstract: The overall goal of this SBIR proposal is to develop and commercialize a new human liver bioassay system for real time, long-term toxicity analysis of chemical agents or drugs in vitro. At present, liver studies are mostly performed using hepatocytes cultured onto synthetic or animal-derived matrices. Unfortunately these models fail to replicate true cell- matrix interactions found in vivo. Moreover, due to phenotypic instability of isolated liver cells there is an urgent need for a long-term culture/assay model. Interestingly, spheroid formation in 3D matrix cultures is shown to improve cell functions. We have recently created a novel human biomatrix culture system- HuBiogel"which supports long-term growth, survival and organization of many cell types. Employing this physiologically-relevant model, we propose to develop new 3D or mini-liver assay platform that exhibit functional benefits and allow precise toxicity prediction. The Phase I specific goals are: 1. Develop and optimize 3D bioassay systems using primary human hepatocytes and defined HuBiogel co- culture configurations;and 2. Evaluate functional and metabolic endpoints with known hepatotoxic agents employing standard microscopy, biochemical and molecular protocols. HuBiogel studies and results will be compared with Collagen I or Matrigel assay systems. In Phase II of this project, hepatotoxicity studies will be expanded to include genomics and proteomics analysis for validation using human liver slice cultures. High throughput adaptability of new bioassay system would allow parallel analysis of liver function, drug metabolism and toxicity profiling. We are confident this advanced in vitro liver toxicity model will positively impact preclinical research and drug discovery arenas. Commercial Importance &Significance: No acceptable commercial liver bioassay model currently exists which utilizes a defined human biomatrix system. VBI will develop new 3D assay formats adaptable to HTS application. Sale of pre-packed culture kits and coated plates would have significant world-wide market, both as a research and diagnostic tool. PUBLIC HEALTH RELEVANCE: Current cell-based assay models for studying drug metabolism &toxicity are of limited utility because liver cells die rapidly in their non-physiologic, 2-dimensional (2D) culture formats. Our company has recently created a novel human biomatrix system- HuBiogel"which supports long term 3-dimensional (3D) growth, survival and organization of a variety of cell types. We propose to employ unique HuBiogel and a NASA-developed rotary culture technology to develop a new 3D human liver bioassay that greatly extends liver cell survival &function beyond current culture methods, providing a much needed tool for real-time analysis of drug metabolism &toxicity in vitro. Cells isolated from donor human livers are the gold standard for pre-clinical evaluation of drug metabolism profiles and potential hepatotoxicity. Unfortunately, these cells lose function and die within hours in traditional cell culturing techniques. The proposed development of human matrix-based 3D bioassay system supporting long-term survival &function of liver cells will have a world-wide market, both as a research and diagnostic tool.